Abstract number: 636
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Abstract number: 636. 24hrs. 12hrs. A. B.

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CONCLUSION

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Conclusion

Abstract number: 636

24hrs

12hrs

A

B

Novel antitumor quinols: the role of thioredoxinEng-Hui Chew, Jenny C. Cookson, Charles S. Matthews, Ji-Hong Zhang, Thilo Hagen, Tracey D. Bradshaw, Malcolm F.G. Stevens, Andrew D. WestwellCentre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, NG7 2RD, UK

Novel antitumor QuinolsAW464 and BW114 exhibit selective antiproliferative activity against colon, renal and breast carcinoma cell lines (GI50 < 0.5 μM). Growth inhibition against renal, colon and breast xenografts has also been demonstrated1, 2. Preliminary results (microarray analysis, insulin reduction assay) suggested that quinols targeta 12 kDa redox-regulating thiol protein thioredoxin (Trx)3. Here we report further evidence of quinols interacting with thioredoxin and the results of investigations into the induction of oxidative stress and apoptosis by quinols.

  • Quinols induce cellular apoptosis

Figure 4: Annexin V-FITC/Propidium Iodide double staining

Propidium Iodide

  • AW464 Binding To Thioredoxin Protein

  • Quinols induce oxidative stress

Figure 1: A, B: Mass spectrum of human Trx

Figure 3: A, B: MTT cell viability

assays (n=4)

Figure 2: ROS production induced by AW464

Annexin V-FITC

Reduced Trx

HCT116 colon cells treated with AW464 (1 µM) showed an increasing proportion of cells undergoing apoptosis between 12 and 24 hours.

Figure 5: Western blot

Dose-dependent PARP cleavage and caspase 3 activation was observed in HCT116 cells treated with AW464 and BW114 for 24 hours.

Cell Number

Campothecin 1 µM, 24hrs (tve ctrl)

AW464

BW114

untreated

1 3 6 0.5 1 3

(µM)

Reduced Trx + AW464

PARP

Cleaved PARP

-actin

H2DCFDA fluorescence

Caspase 3

ROS production was induced in HCT116 colon cells following 7 hours AW464 treatment.

Cleaved Caspase 3

Modulation of the glutathione (GSH) system and quinol cytotoxicity.

- BSO-induced GSH depletion increased cytotoxicity of quinols by 10-fold (Fig3A).

- Supplementation of GSH monoethyl ester

caused a 2-fold reduction in potency (Fig3B).

-actin

No blockade of cytotoxicity by pan caspase inhibitor Z-VAD-FMK and caspase 3 inhibitor Z-DEVD-FMK, suggesting that quinols induce apoptosis via both caspase dependent and independent pathways.

(A) Non-drug treated thioredoxin: mass peak 11,606.

(B) AW464 treated thioredoxin: mass peaks 12,336, 12,586

and 12,821 corresponding to thioredoxin plus 3, 4 and 5

AW464 molecules respectively.

Subsequent MS/MS analysis of trypsin digested peptides

showed additional mass of AW464 only at cysteine residues.

CONCLUSION

We conclude that quinols induce oxidative stress, cellular apoptosis and thioredoxin emerges as a molecular target of these novel antitumor agents.

Reference:

1. Wells et al J. Med. Chem. 2003, 46, 532.

2. Berry et al J. Med.Chem. 2005, 48, 639.

3. Bradshaw et al Cancer Res. 2005, 65, 3911.


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